Vesosomes – hierarchical assemblies consisting of membrane-bound vesicles of various scales – are potentially powerful models of cellular compartmentalization. When a water-in-oil droplet passed through a second lipid monolayer formed in the continuous flow microcentrifuge a bilayer-encapsulated vesosome was created which contained all of the contents of the aqueous phase encapsulated within the vesosome. Encapsulation of nanoscale liposomes within the outer vesosome membrane was confirmed by fluorescence microscopy. Laser diffraction analysis showed that the vesosomes we fabricated were uniform (coefficient of variation of 0.029). The yield of the continuous flow microcentrifuge is high with over 60% of impinging water droplets being converted to vesosomes. Our system provides a fully automatable route for the generation of vesosomes that are able to encapsulate UMI-77 any desired content. The method employed in this work is simple and can be readily applied to a variety of systems providing a facile platform for fabricating multicomponent carriers and model cells. Keywords: Giant unilamellar vesicle Liposome Vesosome 1 Introduction A eukaryotic cell is encapsulated by the plasma membrane which UMI-77 separates the inner and outer environments of the cell . The primary structural scaffolding of the plasma membrane is a phospholipid bilayer. The eukaryotic cell also has several lipid bilayer-encapsulated compartments called organelles including the endoplasmic reticulum (ER) Golgi apparatus lysosomes and peroxisomes. The inner environment and biomolecular contents of each of these organelles differ depending on the specific organelle function. For instance the primary function of the lysosome Flt3 is to degrade proteins. Its inner solution has a low pH and contains 70 varieties of proteases. To protect cytoplasmic proteins from uncontrolled proteolytic degradation a lipid bilayer boundary exists between the interior space and that outside the lysosome. Compartmentalization allows multicompartmentalized cells to regulate the function of each organelle exquisitely facilitating the broad variety of cellular roles observed in eukaryotic biology. To mimic properly the biophysical properties of compartmentalized eukaryotic cells emerging model cell systems must recapitulate this hierarchical compartmentalization i.e. the compartments-within-compartments nature of the eukaryotic cytosol. Various lipid-bilayer structures have been applied as models of cellular compartmentalization. Liposomes are often used as drug carriers and to mimic real cells [2 3 A liposome is surrounded by a phospholipid bilayer that mimics the plasma membrane of real cells. A 10-100 μm UMI-77 liposome is referred to as a giant unilamellar vesicle (GUV). Such structures facilitate direct microscopic observation in real time and GUVs are widely used for research of lipid membrane dynamics mechanics transport and phase behavior . However a GUV is composed of a single UMI-77 membrane that just separates two compartments precluding research and applications of model cells that rely on a multicompartmentalized form. Vesosomes were introduced in 1997 UMI-77 by Zasadzinski and colleagues . Vesosomes are liposome-encapsulated liposomes which widens their applicability as model cells. Unlike the GUVs since the structure is compartmentalized a vesosome can potentially mimic multicompartment behaviors of real cells. Multiple levels of encapsulation also improve the drug delivery properties of vesosomes relative to single-membrane-bounded liposomes. Vesosomes also show improved stability to lipase attack and in serum . Unfortunately GUV-scale vesosome UMI-77 fabrication is complicated by the relatively uncontrolled methods available to form GUVs. GUV fabrication approaches include electroformation  simple hydration  and layer-by-layer emulsification [9 10 Recently GUVs have been fabricated in microfluidic devices [11 12 In particular we have shown the fabrication of asymmetric giant vesicles of uniform size using a microfluidic device . Creating vesosomes usually requires tedious and finely tuned methods and is extremely time-consuming..